Project Abstract Nearly half of newly discovered cancer driver genes discovered by the cancer genomic studies encode proteins involved in histone or DNA modification, including the TET family of DNA dioxygenases. Loss-of-function mutations in TET genes occur early and frequently in human hematopoietic malignancy. Mutations in TET genes, however, are uncommon in solid tumors. Instead, TET activity is significantly reduced in different types of human tumors. We do not know the significance of decreased TET activity in solid tumors. In this study, we hypothesize that TET has a previously unrecognized key function in both the JAK-STAT and NF-?% SDWKZD\V ,QDFWLYDWLRQ RI TET in results in chronic tumor-promoting inflammation and escape from anti-tumor immunity. Thus, stimulating TET activity represents a viable opportunity to enhance antitumor immunity and to improve immunotherapy. We will test this hypothesis by defining the following aspects of TET2 activity: The function and mechanism of TET2 in the JAK-STAT pathway and in tumor immunity (Aim 1); The regulation of TET2 and tumor immunity by reversible monoubiquitylation (Aim 2); and The catalytically independent function and mechanism of TET2 in tumor suppression (Aim 3). During the past funding period, we have made the following discoveries that significantly affect the TET field and that form the foundation for this investigation: (1) Multiple oncometabolites produced or accumulated by mutations in different metabolic enzymes act as antagonists RI ?-NHWRJOXWDUDWH ?.* and inhibit multiple ?.*- dependent enzymes, including TET enzymes. (2) TET activity is dynamically regulated in vivo. (3) Development of solid tumors of many different types is associated with a substantial decrease in TET activity. (4) TET is reversibly monoubiquitylated by CRL4VprBP E3 ligase and UPS15 deubiquitylase, enhancing and impairing TET activity, respectively. This regulation is disrupted by multiple recurrent tumor-derived mutations in TET2. (5) HIV protein Vpr reprograms CRL4VprBP E3 ligase to catalyze polyubiquitylation and degradation of TET proteins to sustain the expression of pro-inflammatory cytokine and promote HIV pathogenesis. (6) Multiple sequence- specific transcription factors (TFs) recruit TET2 to their target genes, including members of NF-?B and STAT families. (7) Loss of TET2 function in tumors impairs interferon signaling, chemokine production, and T cell infiltration, and confers resistance to tumor immunity and immunotherapy. This investigation is built on our pioneering and extensive study of the then newly discovered TET enzymes. It will investigate a novel aspect of cytokine signaling and tumor immunity regulation?by TET- mediated DNA demethylation. It will use newly developed technology and mouse strains to determine how TET proteins regulate gene expression by catalytically-dependent and -independent mechanisms. It will explore a novel regulation of TET by reversible monoubiquitylation and the therapeutic opportunity of this regulation. This investigation represents the first exploration on the function and mechanism of TET in tumor immunity.